Ion dependence of the partially purified mitochondrial dihydropyridine Ca2+ antagonist receptor

The mitochondrial inner membrane contains specific binding sites for dihydropyridine (DHP) Ca2+ antagonists that are associated with an inner mitochondrial membrane anion channel (IMAC) [Mol. Pharmacol. 38:362-369 (1990)]. As in particulate preparations, binding of the DHP (+/-)-[3H]nitrendipine [( 3H]NTR) to partially purified mitochondrial DHP receptors strongly depended on a variety of cations and inorganic as well as organic anions. Monovalent anions saturably stimulated [3H]NTR binding with a potency rank order of I- greater than Br- greater than Cl- greater than F-. The potency rank order for monovalent cations was Cs+ greater than Rb+ greater than Li+ greater than K+ greater than Na+. [3H]NTR binding stimulation potency of the cations strikingly depended on their charge density, with EC50 values being 125 mM for K+, 5 mM for Ca2+, and 41 microM for La3+. This selectivity order clearly differed from one predicted on the basis of a simple surface charge-screening effect of the cations. In general, allosteric ion effects were due to changes in [3H]NTR affinity for the partially purified mitochondrial DHP receptor. SCN- and NO3-, known permeators of the IMAC [J. Biol. Chem. 262:15085-15093 (1987)], stimulated [3H]NTR binding with EC50 values of 26 mM and 96 mM, respectively. The IMAC permeators butylmalonate2- and 1,2,3-benzenetricarboxylate3- were ineffective when given alone but dose-dependently inhibited 500 mM NaCl-stimulated [3H]NTR binding, as did PO4(1.5-) and SO4(2-). Gluconate-, which was reported not to permeate the IMAC, qualitatively behaved as a partial agonist with respect to Cl-. Glucuronate- was without effect on [3H]NTR binding to the partially purified mitochondrial DHP receptor. These results point to the existence of rather large ion-binding domains. The cation-binding site was estimated to have a minimum diameter of 0.67 nm. The anion-binding domain could accommodate either spherical ligands with diameters of up to 0.6 nm or molecules with a flat backbone with dimensions of approximately 0.9 nm x 0.7 nm x 0.3 nm.